Closing Editorial: Immunophenotyping in Autoimmune Diseases and Cancer 3.0.

The mammalian immune system is a Janus-faced network of well-coordinated highly specialized cells and biomolecules [...].

Neural Regeneration in Dry Eye Secondary to Systemic Lupus Erythematosus Is Also Disrupted like in Rheumatoid Arthritis, but in a Progressive Fashion.

Our objective in this study was to analyze the aberrant neural regeneration activity in the cornea by means of in vivo confocal microscopy in systemic lupus erythematosus patients with concurrent dry eye disease. We examined 29 systemic lupus erythematosus patients and 29 age-matched healthy control subjects. Corneal nerve fiber density (CNFD, the number of fibers/mm2) and peripheral Langerhans cell morphology were lower (p < 0.05) in systemic lupus erythematosus patients compared to the control group. Interestingly, corneal nerve branch density, corneal nerve fiber length, corneal nerve fiber total branch density, and corneal nerve fiber area showed a negative correlation with disease duration. A negative correlation was also demonstrated between average corneal nerve fiber density and central Langerhans cell density. This is in line with our hypothesis that corneal somatosensory terminal Piezo2 channelopathy-induced impaired Piezo2-Piezo1 crosstalk not only disrupts regeneration and keeps transcription activated, but could lead to Piezo1 downregulation and cell activation on Langerhans cells when we consider a chronic path. Hence, Piezo2 containing mechanosensory corneal nerves and dendritic Langerhans cells could also be regarded as central players in shaping the ocular surface neuroimmune homeostasis through the Piezo system. Moreover, lost autoimmune neuroinflammation compensation, lost phagocytic self-eating capacity, and lost transcription regulation, not to mention autoantibodies against vascular heparin sulfate proteoglycans and phospholipids, could all contribute to the progressive fashion of dry eye disease in systemic lupus erythematosus.

Evidence of Disruption in Neural Regeneration in Dry Eye Secondary to Rheumatoid Arthritis.

The purpose of our study was to analyze abnormal neural regeneration activity in the cornea through means of confocal microscopy in rheumatoid arthritis patients with concomitant dry eye disease. We examined 40 rheumatoid arthritis patients with variable severity and 44 volunteer age- and gender-matched healthy control subjects. We found that all examined parameters were significantly lower (p < 0.05) in rheumatoid arthritis patients as opposed to the control samples: namely, the number of fibers, the total length of the nerves, the number of branch points on the main fibers and the total nerve-fiber area. We examined further variables, such as age, sex and the duration of rheumatoid arthritis. Interestingly, we could not find a correlation between the above variables and abnormal neural structural changes in the cornea. We interpreted these findings via implementing our hypotheses. Correspondingly, one neuroimmunological link between dry eye and rheumatoid arthritis could be through the chronic Piezo2 channelopathy-induced K2P-TASK1 signaling axis. This could accelerate neuroimmune-induced sensitization on the spinal level in this autoimmune disease, with Langerhans-cell activation in the cornea and theorized downregulated Piezo1 channels in these cells. Even more importantly, suggested principal primary-damage-associated corneal keratocyte activation could be accompanied by upregulation of Piezo1. Both activation processes on the periphery would skew the plasticity of the Th17/Treg ratio, resulting in Th17/Treg imbalance in dry eye, secondary to rheumatoid arthritis. Hence, chronic somatosensory-terminal Piezo2 channelopathy-induced impaired Piezo2-Piezo1 crosstalk could result in a mixed picture of disrupted functional regeneration but upregulated morphological regeneration activity of these somatosensory axons in the cornea, providing the demonstrated abnormal neural corneal morphology.

Disrupted Neural Regeneration in Dry Eye Secondary to Ankylosing Spondylitis-With a Theoretical Link between Piezo2 Channelopathy and Gateway Reflex, WDR Neurons, and Flare-Ups.

This study aimed at analyzing the corneal neural regeneration in ankylosing spondylitis patients using in vivo corneal confocal microscopy in correlation with Langerhans cell density, morphology, and dry eye parameters. Approximately 24 ankylosing spondylitis subjects and 35 age- and gender-matched control subjects were enrolled. Data analysis showed that all corneal nerve-fiber descriptives were lower in the ankylosing spondylitis group, implicating disrupted neural regeneration. Peripheral Langerhans cell density showed a negative correlation with nerve fiber descriptions. A negative correlation between tear film break-up time and corneal nerve fiber total branch density was detected. The potential role of somatosensory terminal Piezo2 channelopathy in the pathogenesis of dry eye disease and ankylosing spondylitis is highlighted in our study, exposing the neuroimmunological link between these diseases. We hypothesized earlier that spinal neuroimmune-induced sensitization due to this somatosensory terminal primary damage could lead to Langerhans cell activation in the cornea, in association with downregulated Piezo1 channels on these cells. This activation could lead to a Th17/Treg imbalance in dry eye secondary to ankylosing spondylitis. Hence, the corneal Piezo2 channelopathy-induced impaired Piezo2-Piezo1 crosstalk could explain the disrupted neural regeneration. Moreover, the translation of our findings highlights the link between Piezo2 channelopathy-induced gateway to pathophysiology and the gateway reflex, not to mention the potential role of spinal wide dynamic range neurons in the evolution of neuropathic pain and the flare-ups in ankylosing spondylitis and dry eye disease.

COVID-19: autoimmunity, multisystemic inflammation and autoimmune rheumatic patients.

Coronavirus disease 2019 (COVID-19) is associated with autoimmunity and systemic inflammation. Patients with autoimmune rheumatic and musculoskeletal disease (RMD) may be at high risk for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In this review, based on evidence from the literature, as well as international scientific recommendations, we review the relationships between COVID-19, autoimmunity and patients with autoimmune RMDs, as well as the basics of a multisystemic inflammatory syndrome associated with COVID-19. We discuss the repurposing of pharmaceutics used to treat RMDs, the principles for the treatment of patients with autoimmune RMDs during the pandemic and the main aspects of vaccination against SARS-CoV-2 in autoimmune RMD patients.

Disease status in human and experimental arthritis, and response to TNF blockade, is associated with MHC class II invariant chain (CD74) isoform expression.

Splice variants of CD74 differentially modulate the activity of cathepsin L (CTSL). As CD74 and CTSL participate in the pathogenesis of inflammatory diseases such as rheumatoid arthritis (RA), we determined whether splice variants of CD74 could be biomarkers of disease activity. Gene expression was measured in mice with collagen-induced arthritis using quantitative PCR (qPCR). In vitro studies using murine macrophage/DC-lineage cells determined the relative influence of macrophage phenotype on isoform expression and the potential to produce CTSL in response to TNF. CD74 splice variants were measured in human RA synovium and RA patients' monocytes. In arthritic mice, the expression of the p41 CD74 isoform was significantly higher in severely affected paws compared with unaffected paws or the paws of naïve mice; the p41 isoform significantly correlated with the expression of TNF in arthritic paws. Compared with M2-like macrophages, M1-like macrophages expressed increased levels of CD74 and had higher expression, secretion and activity of CTSL. RA patients that responded to TNF blockade had significantly higher expression levels of CD74 in circulating monocytes after treatment, compared with non-responders. The expression of the human CD74 isoform a was significantly higher in RA synovia, compared with osteoarthritis synovia, and was associated with CSTL enzymatic activity. This study is the first to demonstrate differential expression of the CD74 p41 isoform in an auto-immune disorder and in response to therapy. The differential expression of CD74 splice variants indicates an association, and potentially a mechanistic role, in the pathogenesis of RA.

Comparison of Homologous and Heterologous Booster SARS-CoV-2 Vaccination in Autoimmune Rheumatic and Musculoskeletal Patients.

Vaccination against SARS-CoV-2 to prevent COVID-19 is highly recommended for immunocompromised patients with autoimmune rheumatic and musculoskeletal diseases (aiRMDs). Little is known about the effect of booster vaccination or infection followed by previously completed two-dose vaccination in aiRMDs. We determined neutralizing anti-SARS-CoV-2 antibody levels and applied flow cytometric immunophenotyping to quantify the SARS-CoV-2 reactive B- and T-cell mediated immunity in aiRMDs receiving homologous or heterologous boosters or acquired infection following vaccination. Patients receiving a heterologous booster had a higher proportion of IgM+ SARS-CoV-2 S+ CD19+CD27+ peripheral memory B-cells in comparison to those who acquired infection. Biologic therapy decreased the number of S+CD19+; S+CD19+CD27+IgG+; and S+CD19+CD27+IgM+ B-cells. The response rate to a booster event in cellular immunity was the highest in the S-, M-, and N-reactive CD4+CD40L+ T-cell subset. Patients with a disease duration of more than 10 years had higher proportions of CD8+TNF-α+ and CD8+IFN-γ+ T-cells in comparison to patients who were diagnosed less than 10 years ago. We detected neutralizing antibodies, S+ reactive peripheral memory B-cells, and five S-, M-, and N-reactive T-cells subsets in our patient cohort showing the importance of booster events. Biologic therapy and <10 years disease duration may confound anti-SARS-CoV-2 specific immunity in aiRMDs.

TLR expression profiles are a function of disease status in rheumatoid arthritis and experimental arthritis.

The role of the innate immune system has been established in the initiation and perpetuation of inflammatory disease, but less attention has been paid to its role in the resolution of inflammation and return to homeostasis. Toll-like receptor (TLR) expression profiles were analysed in tissues with differing disease status in rheumatoid arthritis (RA), ankylosing spondylitis (AS), and in experimental arthritis. TLR gene expression was measured in whole blood and monocytes, before and after TNF blockade. In RA and osteoarthritis synovia, the expression of TLRs was quantified by standard curve qPCR. In addition, four distinct stages of disease were defined and validated in collagen-induced arthritis (CIA), the gold standard animal model for RA - pre-onset, early disease, late disease and immunised mice that were resistant to the development of disease. TLR expression was measured in spleens, lymph nodes, blood cells, liver and the paws (inflamed and unaffected). In RA whole blood, the expression of TLR1, 4 and 6 was significantly reduced by TNF blockade but the differences in TLR expression profiles between responders and non-responders were less pronounced than the differences between RA and AS patients. In RA non-responders, monocytes had greater TLR2 expression prior to therapy compared to responders. The expression of TLR1, 2, 4 and 8 was higher in RA synovium compared to control OA synovium. Circulating cytokine levels in CIA resistant mice were similar to naïve mice, but anti-collagen antibodies were similar to arthritic mice. Distinct profiles of inflammatory gene expression were mapped in paws and organs with differing disease status. TLR expression in arthritic paws tended to be similar in early and late disease, with TLR1 and 2 moderately higher in late disease. TLR expression in unaffected paws varied according to gene and disease status but was generally lower in resistant paws. Disease status-specific profiles of TLR expression were observed in spleens, lymph nodes, blood cells and the liver. Notably, TLR2 expression rose then fell in the transition from naïve to pre-onset to early arthritis. TLR gene expression profiles are strongly associated with disease status. In particular, increased expression in the blood precedes clinical manifestation.

Urine soluble urokinase plasminogen activator receptor as a potential biomarker of lupus nephritis activity.

There is a lack of non-invasive biomarkers to identify lupus nephritis (LN). Soluble urokinase plasminogen activator receptor (suPAR) is a sensitive biomarker of ongoing inflammation and a potential marker of podocyte dysfunction. The aim of this study was to assess urine and plasma suPAR in LN. 14 systemic lupus erythematosus (SLE) patients with newly diagnosed LN, 8 active SLE patients (SLEDAI >8) without LN and 31 healthy individuals were enrolled. Urine and plasma samples were taken before the initiation of LN induction therapy, and monthly thereafter. Global and renal disease activity were defined using the SLEDAI-2K and the SLEDAI-2K renal domain score, respectively. suPAR concentrations were measured with the suPARnostic Flex ELISA assay. Urine and plasma suPAR levels were elevated in SLE patients with active LN compared with resolved LN and healthy controls. Urine suPAR levels were comparable to healthy controls in active SLE without LN. Urine and plasma suPAR levels were higher before than after the initiation of LN induction therapy. Prospective follow-up measurements also suggested that urine suPAR levels raised again in patients with a relapse of LN according to SLEDAI-2K renal domain score, whereas plasma suPAR levels did not correlate with renal disease activity. Urine suPAR is a promising LN activity biomarker, given its isolated elevation in urine in active LN and pronounced decrease with LN improvement.

Investigation of Newly Diagnosed Drug-Naive Patients with Systemic Autoimmune Diseases Revealed the Cleaved Peptide Tyrosine Tyrosine (PYY 3-36) as a Specific Plasma Biomarker of Rheumatoid Arthritis.

There is a current imperative to reveal more precisely the molecular pathways of early onset of systemic autoimmune diseases (SADs). The investigation of newly diagnosed drug-naive SAD patients might contribute to identify novel disease-specific and prognostic markers. The multiplex analysis of 30 plasma proteins in 60 newly diagnosed drug-naive SADs, such as RA (rheumatoid arthritis, n = 31), SLE (systemic lupus erythematosus, n = 19), and SSc (systemic scleroderma, n = 10) patients, versus healthy controls (HCs, n = 40) was addressed. Thirty plasma cytokines were quantified using the Procarta Plex™ panel. The higher expression of IL-12p40, IL-10, IL-13, IFN-γ, M-CSF, IL-4, NTproBNP, IL-17A, BMP-9, PYY (3-36), GITRL, MMP-12, and TNFRSF6 was associated with RA; IL-12p40, M-CSF, IL-4, GITRL, and NTproBNP were higher in SLE; or NTproBNP, PYY (3-36), and MMP-12 were increased in SSc over HCs, respectively. The cleaved peptide tyrosine tyrosine (PYY 3-36) was elevated in RA (361.6 ± 47.7 pg/ml) vs. HCs (163.96 ± 14.5 pg/ml, mean ± SEM, ∗∗∗ p = 4 × 10-5). The CI (95%) was 268.05-455.16 pg/ml for RA vs. 135.55-192.37 pg/ml for HCs. The elevated PYY (3-36) level correlated significantly with the increased IL-4 or GITRL concentration but not with the clinical scores (DAS28, CRP, ESR, RF, aMCV). We are the first to report cleaved PYY (3-36) as a specific plasma marker of therapy-naive RA. Additionally, the multiplex plasma protein analysis supported a disease-specific cytokine pattern in RA, SLE, and SSc, respectively.

Calcium Influx Kinetics and the Characteristics of Potassium Channels in Peripheral T Lymphocytes in Systemic Sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is a chronic, immune-mediated, connective tissue disease causing microvascular abnormalities and fibrosis. The cytoplasmic calcium influx kinetics in T lymphocytes governs lymphocyte activation in this inflammatory process. The inhibition of Kv1.3 and IKCa1 potassium channels reduces calcium influx. METHODS: This study aimed to analyze cytoplasmic calcium influx kinetics following activation in Th1, Th2, and CD8 cells in peripheral blood of 12 healthy individuals and 16 patients with systemic sclerosis using flow cytometry. We also evaluated the effect of the specific inhibition of the Kv1.3 and IKCa1 potassium channels. RESULTS: We observed higher levels of activation in CD8 compared with Th1 cells in SSc. However, the activation of CD8 cells was lower in SSc compared to healthy controls. Moreover, activation of Th1 lymphocytes was slower in SSc than in healthy controls. The inhibition of IKCa1 channels decreased the activation of Th1 cells, while the inhibition of Kv1.3 channels modified the dynamics of activation of Th1 and Th2 lymphocytes in SSc. CONCLUSION: Th1 and CD8 cells demonstrate specific activation dynamics and sensitivity to potassium channel inhibition in SSc, distinguishing this condition both from healthy controls and other autoimmune diseases.

The Impact of Anti-TNF Therapy on CD4+ and CD8+ Cell Subsets in Ankylosing Spondylitis.

OBJECTIVES: Ankylosing spondylitis (AS) is a chronic, progressive immune-mediated inflammatory disease, driven primarily by Th1 and Th17 cells. Anti-TNF therapies are successfully used in AS to achieve and maintain remission. However, their influence on the composition of T-cell subsets is not clear. We aimed to characterize the changes in the T-cell repertoire after a long-term anti-TNF treatment in AS patients. METHODS: Twenty-two AS patients under long-term anti-TNF therapy were evaluated (15 anti-TNF responders and 7 nonresponders). A wide range of cell subtypes was analyzed with flow cytometry and compared with therapy-naïve and short-term data too. RESULTS: Key findings include decreased proportions of naïve CD4 and CD8 cells, increased frequencies of Th1 and Th17 cells and higher Th1/Th2 ratios in the long-term anti-TNF-treated patients (responders, nonresponders and total), which was found to be significant not only when compared with healthy controls, but also with therapy-naïve and short-term anti-TNF-treated AS patients. We noted several alterations within the various activated T-cell subsets - increase in CD4HLADR cells in responders, in CD8HLADR cells in the whole AS group and in responders, and in CD4CD25 cells in responders, and decrease in CD4CD69 cell percentages in long-term treated patients - becoming evident only after long-term anti-TNF therapy. CONCLUSIONS: This study provides a comprehensive assessment of the impact of anti-TNF therapy on the T-cell repertoire in AS. Changes in T-cell phenotype seem to develop progressively during therapy, even in inactive disease, and reflect an ongoing effector T-cell differentiation and activation, along with the parallel compensatory increase in regulatory T cells.

T-Cell Subsets in Rheumatoid Arthritis Patients on Long-Term Anti-TNF or IL-6 Receptor Blocker Therapy.

Data on the impact of biological therapies on the T-cell phenotype in rheumatoid arthritis are limited. Here, we prospectively measured the percentages of 15 circulating T-cell subtypes using flow cytometry. We obtained transversal and longitudinal data in 30 anti-TNF responders, 19 secondary anti-TNF nonresponders, and 43 IL-6R antagonist responders, before, 8 weeks and at least 6 months after biological therapy. Untreated RA patients and healthy controls were also included. The important findings are the following: (1) the proportion of regulatory T-cells (Tregs) which are decreased in untreated RA patients becomes normal in all long-term-treated groups; (2) in anti-TNF responders as well as in nonresponders, the frequencies of naïve CD4+ and CD8+ cells are lower, whereas those of proinflammatory Th1, Th2, and Th17 cells and HLA-DR+-activated cells are higher than those in untreated RA or healthy controls; (3) in IL-6R responders, Th1 proportion is decreased, while that of Th2 and Th17 is increased as compared to that in anti-TNF-treated patients and controls; (4) pending confirmation, a CD4CD69 ratio < 2.43 at baseline, could be useful to predict a good therapeutic response to anti-TNF therapy. This study provides comprehensive information regarding the long-term impacts of those biological therapies on the ecotaxis of T-cells in RA. The ClinicalTrials.gov registration number of our study is NCT03266822.

Alterations of the thrombin generation profile in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disorder resulting in the erosion of the cartilage and bone. Systemic involvement including the cardiovascular system with the risk of atherosclerosis may also occur. Calibrated automated thrombogram (CAT), a commercially available thrombin generation assay is suitable for the general assessment of the functionality of coagulation system. In this study we performed CAT assay in RA patients and in non-affected control subjects (matched for age, sex and comorbidities). Among the CAT parameters Velocity Index increased (from 60 to 83 nM/min), Lag Time and Time to Peak decreased (from 3.47 to 2.83 min and from 6.98 to 5.58 min respectively) in RA. On the other hand, Endogenous Thrombin Potential values decreased (from 1242 to 1108 nM min). The observed alterations were not associated with the applied therapy. These results indicate that the velocity of thrombin formation is increased, while the thrombin generating capability is reduced in RA.

Increased plasma soluble urokinase plasminogen activator receptor levels in systemic sclerosis: possible association with microvascular abnormalities and extent of fibrosis.

BACKGROUND: Urokinase plasminogen activator receptor (uPAR) is a key component of the fibrinolytic system involved in extracellular matrix remodeling and angiogenesis. Novel animal models supported the key role of uPAR not only in fibrosis but also in systemic sclerosis (SSc)-related microvascular abnormalities. The aim of this study was to investigate plasma soluble uPAR (suPAR) levels in SSc, and their association with organ-specific involvement. METHODS: suPAR concentrations were measured by ELISA in SSc patient (n=83) and in healthy controls (n=29). Simultaneously, CRP and ESR were assessed. Detailed clinical data including skin, lung, heart and microvascular characteristics were evaluated at sampling. RESULTS: suPAR values were higher in SSc patients than in controls. Subgroup analysis showed higher suPAR values in diffuse cutaneous- than in limited cutaneous SSc and correlated with anti-Scl-70+. suPAR levels also associated with pulmonary function test parameters of fibrosis, presence of microvascular lesions (e.g., Raynaud phenomenon, naifold capillaroscopic abnormalities and digital ulcers) and arthritis. CONCLUSIONS: Our data indicate that suPAR might be a valuable early diagnostic marker of SSc which also correlates with disease severity.

Comparative single-cell multiplex immunophenotyping of therapy-naive patients with rheumatoid arthritis, systemic sclerosis, and systemic lupus erythematosus shed light on disease-specific composition of the peripheral immune system.

Introduction: Systemic autoimmune diseases (SADs) are a significant burden on the healthcare system. Understanding the complexity of the peripheral immunophenotype in SADs may facilitate the differential diagnosis and identification of potential therapeutic targets. Methods: Single-cell mass cytometric immunophenotyping was performed on peripheral blood mononuclear cells (PBMCs) from healthy controls (HCs) and therapy-naive patients with rheumatoid arthritis (RA), progressive systemic sclerosis (SSc), and systemic lupus erythematosus (SLE). Immunophenotyping was performed on 15,387,165 CD45+ live single cells from 52 participants (13 cases/group), using an antibody panel to detect 34 markers. Results: Using the t-SNE (t-distributed stochastic neighbor embedding) algorithm, the following 17 main immune cell types were determined: CD4+/CD57- T cells, CD4+/CD57+ T cells, CD8+/CD161- T cells, CD8+/CD161+/CD28+ T cells, CD8dim T cells, CD3+/CD4-/CD8- T cells, TCRγ/δ T cells, CD4+ NKT cells, CD8+ NKT cells, classic NK cells, CD56dim/CD98dim cells, B cells, plasmablasts, monocytes, CD11cdim/CD172dim cells, myeloid dendritic cells (mDCs), and plasmacytoid dendritic cells (pDCs). Seven of the 17 main cell types exhibited statistically significant frequencies in the investigated groups. The expression levels of the 34 markers in the main populations were compared between HCs and SADs. In summary, 59 scatter plots showed significant differences in the expression intensities between at least two groups. Next, each immune cell population was divided into subpopulations (metaclusters) using the FlowSOM (self-organizing map) algorithm. Finally, 121 metaclusters (MCs) of the 10 main immune cell populations were found to have significant differences to classify diseases. The single-cell T-cell heterogeneity represented 64MCs based on the expression of 34 markers, and the frequency of 23 MCs differed significantly between at least twoconditions. The CD3- non-T-cell compartment contained 57 MCs with 17 MCs differentiating at least two investigated groups. In summary, we are the first to demonstrate the complexity of the immunophenotype of 34 markers over 15 million single cells in HCs vs. therapy-naive patients with RA, SSc, and SLE. Disease specific population frequencies or expression patterns of peripheral immune cells provide a single-cell data resource to the scientific community.

Analysis by flow cytometry of calcium influx kinetics in peripheral lymphocytes of patients with rheumatoid arthritis.

The transient increase of the cytoplasmic free calcium level in T lymphocytes plays a key role in initiating and maintaining the autoimmune reaction in rheumatoid arthritis (RA). Kv1.3 and IKCa1 potassium channels are important regulators of the maintenance of calcium influx during lymphocyte activation and present a possible target for selective immunomodulation. We aimed to compare peripheral T lymphocyte calcium influx kinetics upon activation in patients with recently diagnosed and established RA, and to demonstrate the differences in analysis of kinetic flow cytometry data when using two different algorithms. We took peripheral blood samples from nine patients with recently diagnosed and six patients with established RA. We evaluated calcium influx kinetics following activation in CD4, Th1, Th2, and CD8 cells applying an approach based on smoothing of median fluorescence values (FlowJo) and an algorithm based on function fitting (FacsKin). We assessed the sensitivity of the above subsets to specific inhibition of the Kv1.3 and IKCa1 potassium channels. Th2 cells of patients with established RA react slower to activating stimuli, whereas CD8 cells show a faster reaction than in patients with recently diagnosed RA. While initially Th1 cells are less sensitive to the inhibition of Kv1.3 and IKCa1 channels in RA, their sensitivity increases along with the duration of the disease. With the algorithm of function fitting instead of smoothing, more statistically significant differences of potassium channel inhibition between the two RA groups could be demonstrated. The function fitting algorithm applied by FacsKin is suitable to provide a common basis for evaluating and comparing flow cytometry kinetic data.

Neuroimmune interactions in Sjögren's syndrome: relationship of exocrine gland dysfunction with autoantibodies to muscarinic acetylcholine receptor-3 and mental health status parameters.

OBJECTIVES: Antimuscarinic acetylcholine receptor-3 (m3AChR) autoantibodies have been described in primary Sjögren's syndrome (pSS). The aim of this study was to compare various methods for their detection and to assess the contributions of anti-m3AChR and other immunological and psychosocial factors to the pathomechanism of secondary SS (sSS). METHODS: Sixty-five rheumatoid arthritis (RA) patients, 103 systemic lupus erythematosus (SLE) patients, 76 pSS patients and 50 controls were compared. Three immunodominant epitopes of m3AChR were synthesized and used in ELISA. Two extracellular epitopes were also prepared in fusion with glutathione-S-transferase and one in conjugation with bovine serum albumin. Mental health status was assessed with the 36-item Short-Form Health Survey and Functional Assessment of Chronic Illness Therapy fatigue scale. Correlations were evaluated between glandular function and anti-m3AChR positivities and specificities, features of SLE and RA, and mental health parameters. RESULTS: Fourteen RA and 27 SLE patients had sSS. The autoantibody levels to all epitopes of m3AChR were significantly higher in pSS and SLE patients than in the controls. The fusion protein forms discriminated RA from pSS and SLE; furthermore, the YNIP fusion protein also distinguished pSS from SLE. The prevalence and the mean levels of all autoantibodies did not differ statistically between sicca and non-sicca SLE or RA patients. Glandular dysfunction correlated with higher age in SLE and RA and an impaired health-related quality of life in SLE. CONCLUSIONS: The second and third extracellular loops of m3AChR are antigenic in pSS. Immunoassays with antigens as fusion peptides demonstrate the best performance. Sicca SLE patients have worse mental health status. Anti-m3AChR antibodies represent a peculiar example of neuroimmune interactions.

Soluble urokinase plasminogen activator receptor (suPAR) in the assessment of inflammatory activity of rheumatoid arthritis patients in remission.

BACKGROUND: Soluble urokinase plasminogen activator receptor (suPAR) is a biomarker increasingly used for the assessment of systemic inflammation. We aimed to evaluate suPAR for the assessment of inflammatory activity in rheumatoid arthritis (RA) patients in remission. METHODS: In our cross-sectional study we measured plasma suPAR and C-reactive protein (CRP) levels as well as erythrocyte sedimentation rate (ESR) in 120 RA patients at various stages of disease activity and 29 healthy age-matched controls. RESULTS: suPAR, CRP and ESR values were higher in RA patients compared to healthy individuals. When suPAR levels were analyzed according to DAS28 scores of RA patients, suPAR level in the subgroup with DAS28≤2.6 was lower than in the subgroup with DAS28>2.6, but still higher than in controls [4.45 (3.33-5.56) ng/mL vs. 3.66 (3.10-4.67) ng/mL vs. 2.80 (2.06-3.42) ng/mL, p<0.0001, median (interquartile range)]. In contrast, CRP and ESR values were comparable in the subgroup with DAS28≤2.6 and in healthy individuals. We further analyzed the correlation between the number of tender and/or swollen joints and suPAR levels in RA patients in remission. suPAR values were significantly higher in patients with four tender and/or swollen joints than in patients with 2-3 or 0-1 tender and/or swollen joints. CONCLUSIONS: While CRP and ESR values indicate remission of the chronic inflammatory process in RA, suPAR values are still elevated compared to healthy individuals. suPAR might be particularly valuable in the recognition of inflammatory activity in patients who are in remission according to DAS28 scores but have symptoms of tender and/or swollen joints.

Comparison of humoral and cellular immune responses in hematologic diseases following completed vaccination protocol with BBIBP-CorV, or AZD1222, or BNT162b2 vaccines against SARS-CoV-2.

Background: Vaccination has proven the potential to control the COVID-19 pandemic worldwide. Although recent evidence suggests a poor humoral response against SARS-CoV-2 in vaccinated hematological disease (HD) patients, data on vaccination in these patients is limited with the comparison of mRNA-based, vector-based or inactivated virus-based vaccines. Methods: Forty-nine HD patients and 46 healthy controls (HCs) were enrolled who received two-doses complete vaccination with BNT162b2, or AZD1222, or BBIBP-CorV, respectively. The antibodies reactive to the receptor binding domain of spike protein of SARS-CoV-2 were assayed by Siemens ADVIA Centaur assay. The reactive cellular immunity was assayed by flow cytometry. The PBMCs were reactivated with SARS-CoV-2 antigens and the production of activation-induced markers (TNF-α, IFN-γ, CD40L) was measured in CD4+ or CD8+ T-cells ex vivo. Results: The anti-RBD IgG level was the highest upon BNT162b2 vaccination in HDs (1264 BAU/mL) vs. HCs (1325 BAU/mL) among the studied groups. The BBIBP-CorV vaccination in HDs (339.8 BAU/mL ***p < 0.001) and AZD1222 in HDs (669.9 BAU/mL *p < 0.05) resulted in weaker antibody response vs. BNT162b2 in HCs. The response rate of IgG production of HC vs. HD patients above the diagnostic cut-off value was 100% vs. 72% for the mRNA-based BNT162b2 vaccine; 93% vs. 56% for the vector-based AZD1222, or 69% vs. 33% for the inactivated vaccine BBIBP-CorV, respectively. Cases that underwent the anti-CD20 therapy resulted in significantly weaker (**p < 0.01) anti-RBD IgG level (302 BAU/mL) than without CD20 blocking in the HD group (928 BAU/mL). The response rates of CD4+ TNF-α+, CD4+ IFN-γ+, or CD4+ CD40L+ cases were lower in HDs vs. HCs in all vaccine groups. However, the BBIBP-CorV vaccine resulted the highest CD4+ TNF-α and CD4+ IFN-γ+ T-cell mediated immunity in the HD group. Conclusion: We have demonstrated a significant weaker overall response to vaccines in the immunologically impaired HD population vs. HCs regardless of vaccine type. Although, the humoral immune activity against SARS-CoV-2 can be highly evoked by mRNA-based BNT162b2 vaccination compared to vector-based AZD1222 vaccine, or inactivated virus vaccine BBIBP-CorV, whereas the CD4+ T-cell mediated cellular activity was highest in HDs vaccinated with BBIBP-CorV.